Abstract While food allergy has emerged as a major public health issue that affects large numbers of individuals, the mechanisms underlying oral tolerance breakdown in food allergy have remained elusive. Studies have shown that effective oral immunotherapy that promotes tolerance in previously food allergic individuals is associated with elicitation of regulatory T cell (TR) responses. Furthermore, we have demonstrated that whereas TR cells play a requisite role in enforcing oral tolerance to food antigens, their regulatory function becomes impaired in food allergy. Specifically, we have found that allergen-specific TR cells in food allergic mice and humans acquire a Th2 cell-like transcriptional program and cytokine expression profile, and have shown this pathogenic reprogramming of TR cells to be instrumental in driving Th2 immune responses in food allergy. Accordingly, elucidating the mechanisms by which Th2 cell-like TR cells promote the acquisition of food allergy is of paramount interest in developing a unified, testable model of oral tolerance breakdown and devising interventions to obviate it. Our preliminary mouse studies have shown that TR cell-derived TGF-?1 plays an essential role in regulating mucosal immune responses to food antigens, and that its levels drop by about 50% in Th2 cell-like programmed TR cells. Significantly, TR cell-specific Tgfb1 haplo-insufficiency predisposes mice to food allergy, thus confirming the functional significance of decreased TGF-?1 expression in Th2 cell-like reprogrammed TR cells. The overarching hypothesis of this proposal is that TR cell-derived TGF-?1 plays a pivotal, non-redundant role in the suppression of Th2 cell immunity and, reciprocally, that the acquisition by TR cells of a Th2 cell-like genetic program acts to promote food allergy, at least in part, by suppressing Tgfb1 expression. The specific aims of this proposal will mechanistically establish the link between genetic and acquired TR cell-specific TGF-?1 deficiency with the development of food allergy. The experiments under Aim 1 will explore TR cell TGF-?1-deficiency as a key mechanism that perpetuates chronic Th2 cell inflammation, while Aim 2 will investigate molecular mechanisms by which IL-4/STAT6 signaling regulates the Tgfb1 locus in TR cells. Aim 3 seeks to validate that Th2 cell-like TR cells in human allergic subjects are deficient in TGF-?1 production, and that their TGF-?1 expression and functional competency can be restored by antagonism of their Th2-cell like program. The proposed studies will help identify fundamental mechanisms operative in oral tolerance breakdown in food allergy and offer promising interventional strategies to re-establish tolerance.